Polyhydroxyalkanoate synthase and gene encoding the same enzyme

ABSTRACT

A novel polyhydroxyalkanoate (PHA) synthase derived from a microorganism capable of producing a PHA having a novel side-chain structure and a DNA encoding the amino acid sequence for the synthase are provided. Two PHA synthase proteins (SEQ ID Nos. 1 and 3) derived from  Pseudomonas jessenii  P161 (FERM BP-7376) and PHA synthase genes encoding these PHA synthases are provided, respectively (SEQ ID Nos. 2 and 4). A recombinant microorganism is endowed with a PHA producing ability.

This is a continuation-in-part application of U.S. patent applicationSer. No. 10/218,519 filed on Aug. 15, 2002, which is a divisionapplication of U.S. application Ser. No. 09/821,016 filed on Mar. 30,2001 now U.S. Pat. No. 6,485,951.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a polyhydroxyalkanoate (hereinafter, referredto as a “PHA”) synthase, a gene encoding the PHA synthase, a recombinantvector containing the gene, a transformant capable of expressing the PHAsynthase which has been transformed by the recombinant vector, a processfor producing the PHA synthase utilizing the transformant, and a processfor preparing the PHA utilizing the transformant. In particular, thisinvention relates to a microorganism-derived PHA synthase capable ofproducing a polyhydroxyalkanoate and a gene encoding the PHA synthaseutilized for expressing the PHA synthase by transformation.

2. Related Background Art

There have been reported a number of microorganisms producingpoly-3-hydroxybutyric acid (PHB) or another PHA and storing it therein(“Biodegradable Plastic Handbook”, edited by Biodegradative PlasticResearch Society, NTS Co. Ltd., p. 178-197). These polymers may be, asconventional plastics, used for producing a variety of products by, forexample, melt-processing. Since they are biodegradable, they have anadvantage that they can be completely degraded by microorganisms in thenatural environment, and they do not cause pollution due to remaining inthe natural environment like many conventional polymer compounds.Furthermore, they are excellently biocompatible, and thus are expectedto be used in applications such as a medical soft member.

It is known that a composition and a structure of such a PHA produced bya microorganism may considerably vary depending on the type of amicroorganism used for the production, a culture-medium composition andculturing conditions. Investigations have been, therefore, mainlyfocused on controlling such a composition or structure for the purposeof improving physical properties of a PHA.

For example, Japanese Patent Application Nos. 7-14352 and 8-19227 andJapanese Examined Publication No. 6-15604 describe that Alcaligeneseutropus H16 (ATCC No. 17699) and its variants may produce3-hydroxybutyric acid (3HB) and its copolymer with 3-hydroxyvaleric acid(3HB) with various composition ratios by changing a carbon source duringculturing.

Japanese Patent Publication No. 2642937 discloses that PHA in which amonomer unit is 3-hydroxyalkanoate with 6 to 12 carbon atoms may beproduced by supplying a non-cyclic aliphatic hydrocarbon as a carbonsource to Pseudomonas oleovorans (ATCC No. 29347).

Japanese Patent Application Laid-Open No. 5-7492 discloses methods inwhich Methylobaterium sp., Paracoccus sp., Alcaligenes sp., andPseudomonas sp. are contacted with a primary alcohol with 3 to 7 carbonatoms to produce a copolymer of 3HB and 3HV.

Japanese Patent Application Laid-Open Nos. 5-93049 and 7-265065 disclosethat Aeromonas caviae is cultured using oleic acid or olive oil as acarbon source to produce a two-component copolymer of 3HB and3-hydroxyhexanoic acid (3HHx).

Japanese Patent Application Laid-Open No. 9-191893 discloses thatComamonas acidovorans IF013852 is cultured using gluconic acid and1,4-butanediol as carbon sources to produce a polyester having 3HB and4-hydroxybutyric acid as monomer units.

Furthermore, it is reported that certain microorganisms produce PHAshaving a variety of substituents such as unsaturated hydrocarbon, ester,aryl (aromatic), and cyano groups, halogenated hydrocarbon and epoxide.Recently, there have been attempts for improving physical properties ofa PHA produced by a microorganism using such a procedure. For example,Makromol. Chem., 191, 1957-1965 (1990); Macromolecules, 24, 5256-5260(1991); and Chirality, 3, 492-494 (1991) describe production of a PHAcomprising 3-hydroxy-5-phenylvaleric acid (3HPV) as a monomer unit byPseudomonas oleovorans, where variations in polymer physical propertiesprobably due to the presence of 3HPV were observed.

As described above, microorganism-produced PHAs with variouscombinations of composition and structure have been obtained by varyingfactors such as the type of a microorganism used, a culture mediumcomposition and culturing conditions. Each microorganism has anintrinsic PHA synthase with a substrate specificity which issignificantly different from others. Thus, it has been difficult toproduce PHAs comprising different monomer units suitable to a variety ofapplications using known microorganisms or PHA synthases in such knownmicroorganisms.

Meanwhile, as described above, a PHA having a variety of substituents inits side chains may be expected to be a “functional polymer” havingsignificantly useful functions and properties owing to the properties ofthe introduced substituents. It is, therefore, extremely useful andimportant to search and develop a microorganism which can produce andstore a very useful polymer having both such functionality andbiodegradability. Furthermore, identification of a PHA synthase involvedin production of the highly useful PHA and obtaining a gene encoding thePHA synthase may allow us to produce a novel transformed microorganismcapable of producing a desired PHA. That is, constructing a recombinantvector comprising a gene encoding a PHA synthase and providing amicroorganism transformed by the recombinant vector may allow us toprepare a PHA using the transformed microorganism or to express arecombinant type of PHA synthase. As described above, it may beimportant that a transformed microorganism is used to prepare a desiredPHA for providing a highly useful tool for improving a productivity forthe PHA and for promoting utilization of the PHA.

SUMMARY OF THE INVENTION

Objects of this invention which can solve the above problems are tosearch a novel microorganism capable of producing and storing inmicroorganisms a PHA having a novel side-chain structure, to identify anenzyme protein related to the ability of producing the novel PHA, i.e.,a novel PHA synthase, and to determine a gene encoding its amino acidsequence. More specifically, an object of the present invention is toprovide a novel PHA synthase derived from a microorganism producing aPHA having a novel side chain structure and a DNA encoding its aminoacid sequence. Another object of this invention is to provide arecombinant vector to which a DNA encoding an available PHA synthase isintroduced and which is used for transformation of a microorganism and atransformed microorganism produced using the recombinant vector. Afurther object of this invention is to provide a process for expressingand producing a recombinant PHA synthase in the transformedmicroorganism and a process for preparing a desired PHA using thetransformed microorganism.

Still another object of this invention is to provide a modified PHAsynthase in which its amino acid sequence is modified as long as anenzyme activity is not affected in expression of the recombinant PHAsynthase in the transformed microorganism as described above and a DNAencoding the modified amino acid sequence.

For developing a PHA having a novel side-chain structure useful as, forexample, a device material or a medical material aiming at solving theabove problems, the inventors have searched a novel microorganismcapable of producing and storing the desired PHA therein. Additionally,the inventors have intensely investigated selected novel microorganismsproducing a novel PHA for identifying a PHA synthase involved inproduction of the novel PHA and for obtaining a gene encoding the PHAsynthase. Furthermore, the inventors have conducted investigation forconstructing a recombinant vector with a gene for the obtained PHAsynthase, transforming a host microorganism using the recombinantvector, expressing a recombinant PHA synthase in the transformedmicroorganism obtained and determining production of the desired PHA.

In the course of the above investigation, the inventors synthesized5-(4-fluorophenyl) valeric acid (FPVA) represented by formula (II):

and separated from a soil a novel microorganism capable of convertingthe above compound (II) as a starting material (substrate) intocorresponding 3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV)represented by formula (III):

and producing and storing a novel PHA with a monomer unit represented byformula (I):

derived from 3HFPV. The novel microorganism separated is designated asP161 strain. The inventors have also found that in addition to the aboveenzymatic activity for converting FPVA into 3HFPV, the P161 strain mayalso use 4-phenoxybutyric acid (PxBA) represented by formula (IV):

as a starting material (substrate) to convert it into3-hydroxy-4-phenoxybutyric acid (3HPxB) represented by formula (V):

and to produce and store a PHA with a monomer unit represented byformula (VI):

derived from 3HPxB. There have been no reports for microbial productionof a PHA comprising 3HPxB as a monomer unit using PxBA as a substrate orfor microbial production of a PHA comprising 3HPxB as a solephenoxy-containing monomer unit.

An example of a microorganism capable of producing and storing a PHAwith a monomer unit represented by formula (VI) derived from 3HPxB usinga substrate other than PxBA is Pseudomonas oleovorans using8-phenoxyoctanoic acid (PxOA) as a substrate described inMacromolecules, 29, 3432-3435, 1996. In Pseudomonas oleovorans,8-phenoxyoctanoic acid (PxOA) is used as a substrate, which is totallydifferent from the enzymatic reaction in P161 strain where PxBA is usedas a substrate to produce a PHA with a monomer unit represented byformula (VI) derived from a corresponding 3HPxB. In addition, for thecomposition of a PHA produced, the reported process using Pseudomonasoleovorans provides a copolymer consisting of three monomer units, i.e.,3-hydroxy-8-phenoxyoctanoic acid corresponding to PxOA as a substrate,3-hydroxy-6-phenoxyhexanoic acid as a byproduct derived from ametabolite of the substrate, and the desired 3HPxB. On the other hand, aprocess where P161 strain acts on the substrate PxBA provides a PHA with3HPxB derived from PxBA as a sole phenoxy-containing monomer unit.Taking the compositions of the PHAs also into consideration, it seemsthat there is fundamental difference in substrate specificity of a PHAsynthase between Pseudomonas oleovorans used in the above processreported and P161 strain. That is, a PHA synthase produced by P161strain is more preferable for production of a PHA with 3HPxB as amonomer unit.

Furthermore, the inventors have found that P161 strain can use6-phenylhexanoic acid (PHxA) represented by formula (VII):

as a starting material (substrate) to convert it into corresponding3-hydroxy-6-phenylhexanoic acid (3HPHx) represented by formula (VIII):

and to produce and store a novel PHA with a monomer unit represented byformula (IX):

derived from 3HPHx.

Microbiological Properties of P161 strain are as follows.

<Microbiological Properties of P161 Strain>

Morphologic Properties

Cell shape and size: Sphere, φ0.6 μm

Bacilliform, 0.6 μm×1.5 to 2.0 μm

Cell polymorphism: Yes (elongation)

Motility: Yes

Sporulation: No

Gram stainability: Negative

Colonization: Circular, smooth in the overall

periphery, low convex, smooth surface, pale yellow

Physiological properties

Catalase: Positive

Oxidase: Positive

O/F test: oxidized form

Reduction of a nitrate: Positive

Indole formation: Negative

Acidification of dextrose: Negative

Arginine dihydrolase: Positive

Urease: Negative

Esculin hydrolysis: Negative

Gelatin hydrolysis: Negative

β-Galactosidase: Negative

Fluorochrome production on King's B agar: Positive

Substrate Assimilation Ability

Dextrose: Positive

L-Arabinose: Positive

D-Mannose: Positive

D-Mannitol: Positive

N-Acetyl-D-glucosamine: Positive

Maltose: Negative

Potassium gluconate: Positive

n-Capric acid: Positive

Adipic acid: Negative

dl-Malic acid: Positive

Sodium citrate: Positive

Phenyl acetate: Positive

From these microbiological properties, the inventors have attempted tocategorize P161 strain according to Bergey's Manual of SystematicBacteriology, Volume 1 (1984) and Bergey's Manual of DeterminativeBacteriology 9th ed. (1994) to determine that the strain belongs toPseudomonas sp. Its taxonomic position could not been determined fromthese microbiological properties.

Thus, for categorizing P161 strain from its genetic properties, theinventors sequenced its 16S rRNA (SEQ ID NO. 5) and compared itshomology with the sequence of a 16S rRNA in a known Pseudomonas sp.microorganism. The results indicate quite higher homology in a 16S rRNAsequence between P161 strain and a known Pseudomonas jessenii.Furthermore, microbiological properties described for the knownPseudomonas jessenji in System. Appl. Microbiol., 20, 137-149 (1997) andSystem. Appl. Microbiol., 22, 45-58 (1999) was compared with those forP161 strain and observed considerable homology. From these results, itwas judged to be proper to categorize P161 strain in Pseudomonasjessenii, and thus it is designated as Pseudomonas jessenii P161. Therehave been no reports on a strain in Pseudomonas jessenii capable ofproducing a PHA as exhibited by P161 strain. The inventors have,therefore, determined that P161 strain is a novel microorganism. Theapplicant deposited Pseudomonas jessenii P161 to Patent MicroorganismDepository Center in the National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology, Ministry ofInternational Trade and Industry, under the deposition number of FERMP-17445. P161 strain has been internationally deposited on the basis ofthe Budapest Treaty, and its international accession number is “FERMBP-7376”.

The inventors achieved cloning a gene for a PHA synthase from the novelmicroorganism P161 strain and sequenced the gene. The inventors alsodetermined an amino acid sequence for the PHA synthase encoded by thegene. Based on the above observation, the present invention wasachieved.

Specifically, a PHA synthase of the present invention is apolyhydroxyalkanoate synthase having an amino acid sequence of SEQ IDNO. 1 or 3. Furthermore, the PHA synthase of the present invention maybe a PHA synthase substantially retaining the amino acid sequence of SEQID NO. 1 and having a modified amino acid sequence where amino acids aredeleted, substituted or added as long as it does not deteriorate anactivity as the polyhydroxyalkanoate synthase, or a PHA synthasesubstantially retaining the amino acid sequence of SEQ ID NO. 3 andhaving a modified amino acid sequence where amino acids are deleted,substituted or added as long as it does not deteriorate activity as thepolyhydroxyalkanoate synthase.

A PHA synthase gene of the present invention is a gene for apolyhydroxyalkanoate synthase comprising a DNA encoding the amino acidsequence of SEQ ID NO. 1 or the sequence of its modified amino acid, ora gene for a polyhydroxyalkanoate synthase comprising a DNA encoding theamino acid sequence of SEQ ID NO. 3 or the sequence of its modifiedamino acid. Embodiments of a PHA synthase gene of the present inventionderived from a genome gene in P161 strain include a PHA synthase genecomprising a DNA sequence of SEQ ID NO. 2 as a DNA encoding the aminoacid sequence of SEQ ID NO. 1 and a PHA synthase gene comprising a DNAsequence of SEQ ID NO. 4 as a DNA encoding the amino acid sequence ofSEQ ID NO. 3.

This invention also provides a recombinant vector comprising a gene DNAencoding the above amino acid sequence as a polyhydroxyalkanoatesynthase gene. This invention also provides a transformed microorganismtransformed by introducing a recombinant vector adapted to a host.

The present invention also provides a process for preparing apolyhydroxyalkanoate comprising the steps of culturing the transformedmicroorganism to which a recombinant vector has been introduced in aculture medium containing a substrate for a polyhydroxyalkanoatesynthase and collecting the polyhydroxyalkanoate from the culturepreparation. The present invention also provides a process for producinga polyhydroxyalkanoate comprising the steps of culturing the transformedmicroorganism to which a recombinant vector has been introduced andmaking the transformed microorganism produce the polyhydroxyalkanoate.

A preferable process for producing a polyhydroxyalkanoate may utilizesubstrate specificity characteristic of a polyhydroxyalkanoate synthasederived from P161 strain: for example, preparation of apolyhydroxyalkanoate comprising a monomer unit represented by formula(I) derived from 3HFPV utilizing the above transformed microorganism;preparation of a polyhydroxyalkanoate comprising a monomer unitrepresented by formula (VI) derived from 3HPxB, or preparation of apolyhydroxyalkanoate comprising a monomer unit represented by formula(IX) derived from 3HPHx.

A PHA synthase and a gene encoding the PHA synthase of the presentinvention are derived from a novel microorganism, Pseudomonas jesseniiP161 strain, and exhibit such substrate specificity that it selectivelyproduces a PHA comprising a monomer unit having a novel side chainstructure. A recombinant vector comprising the PHA synthase gene and amicroorganism transformed by the recombinant vector are capable ofproducing a PHA exhibiting substrate specificity similar to Pseudomonasjessenii P161. Thus, a PHA synthase gene of this invention encodes anenzyme which permits preparation of a PHA selectively comprising amonomer unit having a novel side-chain structure and allows us to createa transformed microorganism useful for preparing a PHA having varioususeful physical properties which may be expected to be applied to afunctional polymer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A PHA synthase of this invention is an enzyme protein derived from anovel microorganism isolated by the present inventors, Pseudomonasjessenii P161 (FERM BP-7376). Specifically, it can convert5-(4-fluorophenyl)valeric acid (FPVA) into corresponding3-hydroxy-5-(4-fluorophenyl)valeric acid (3HFPV), 4-phenoxybutyric acid(PxBA) into corresponding 3-hydroxy-4-phenoxybutyric acid (3HPxB) or6-phenylhexanoic acid (PHxA) into corresponding3-hydroxy-6-phenylhexanoic acid (3HPHx) and thus has enzymatic activityinvolved in production of a PHA comprising a corresponding monomer unit.

A PHA synthase and a gene encoding the enzyme of this invention will bemore specifically described.

From P161 strain, the inventors have cloned a gene translated into a PHAsynthase which exhibits the above substrate specificity, to determinethe presence of a PHA synthase comprising at least two amino acidsequences. Specifically, a PHA synthase of this invention in achromogene in P161 strain comprises two enzymes, i.e., a PHA synthasecomprising the amino acid sequence of SEQ ID NO. 1 encoded by a DNAhaving the sequence of SEQ ID NO. 2 and a PHA synthase comprising theamino acid sequence of SEQ ID NO. 3 encoded by a DNA having the sequenceof SEQ ID NO. 4. Gene DNAs of the sequences of SEQ ID NOs. 2 and 4 maybe cloned by the following procedure.

Since a PHA synthase is an enzyme protein translated from a chromogene,a chromosome DNA containing a desired PHA synthase is first obtained. Achromosome DNA may be separated from P161 strain cells by a knownseparation method. For example, P161 strain is cultured in a LB mediumor an M9 medium supplemented with an appropriate carbon source, disruptand treated as described by, for example, Marmer et al. in Journal ofMolecular Biology, Vol. 3, p. 208 (1961) to prepare a chromosome DNA.

Then, a gene library is prepared from the chromosome DNA thus obtained.The chromosome DNA is degraded using an appropriate restriction enzyme(e.g., Sau3AI) and a fragment with a proper length is ligated with aligatable vector truncated with a restriction enzyme (e.g., BamHI) toprepare a gene library.

Depending on a vector used in preparing a library, a proper fragmentlength varies, e.g., about 4000 to 25000 bps for a usual plasmid vectorand about 15000 to 30000 bps for a cosmid or phage vector. A properlength of DNA fragment may be collected by a known method such as amethod using a sucrose density gradient or using an agarose geldescribed in Molecular Cloning, Cold Spring Harbor Laboratory (1982).

Since E. coli is used as a host microorganism in a gene library, avector is a phage vector or plasmid vector which can autonomously growin the host microorganism (E. coli). Examples of phage or cosmic vectorsgenerally used include pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII,λZAPII, λgt10, λgt11, Charon4A and Charon21A. Examples of frequentlyused plasmid vectors include pBR, pUC, pBluescriptll, pGEM, pTZ and pETgroups. In addition to E. coli, various shuttle vectors may be used,e.g., vectors which may autonomously grow in a plurality of hostmicroorganisms such as Pseudomonas sp. Again, these vectors may be,depending on a chromosome DNA to be ligated to them, truncated with aproper restriction enzyme to provide a desired fragment.

A chromosome DNA fragment may be ligated with a vector fragment using aDNA ligase. For example, a commercially available ligation kit (TakaraShuzo Co., Ltd., etc.) may be used. Thus, for example, variouschromosome DNA fragments may be ligated with a plasmid vector fragmentto prepare a mixture of recombinant plasmids comprising various DNAfragments (hereinafter, referred to as a “gene library”).

In addition to a method using a proper length of chromosome DNAfragment, a gene library may be prepared by a method that all mRNAs areextracted from P161 strain, purified and used for preparation of a cDNAfragment using a reverse transcriptase as described in MolecularCloning, Cold Spring Harbor Laboratory, 1982. Alternatively, a preparedvector is used in a gene library to transform or transduce to E. coli,and then the host E. coli is cultured to amplify the gene library to alarge amount as described in Molecular Cloning, Cold Spring HarborLaboratory, 1982.

A recombinant vector comprising a gene DNA fragment may be introducedinto a host microorganism by a known method. For example, when using E.coli as a host microorganism, a recombinant plasmid vector may beintroduced using a calcium chloride method (Journal of MolecularBiology, Vol. 53, p. 159 (1970)), a rubidium chloride method (Methods inEnzymology, Vol. 68, p. 253 (1979)), electroporation (Current Protocolsin Molecular Biology, Vol. 1, p. 1.8.4 (1994)). When using a cosmidvector or phage vector, transduction in a host E. coli may be conductedusing in vitro packaging (Current Protocols in Molecular Biology, Vol.1, p. 5.7.1 (1994)). Alternatively, conjugational transfer with a strainretaining a recombinant vector may be utilized to prepare a strainretaining a vector.

Then, from the gene library, a probe is prepared for obtaining a DNAfragment comprising a PHA synthase gene of P161 strain.

Some base sequences have been reported for PHA synthase genes in knownmicroorganisms; for example, Peoples, O. P. and Sinskey, A. J., J. Biol.Chem., 264, 15293 (1989); Huisman, G. W. et al., J. Biol. Chem., 266,2191 (1991); Pieper, U. et al., FEMS Microbiol. Lett., 96, 73 (1992);Timm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15(1992);Matsusaki, H. et al., J. Bacteriol., 180, 6459 (1998). These reportedsequences are compared to select a region where a sequence is preservedto a higher degree and thus to design an oligonucleotide for a primerused in polymerase chain reaction (hereinafter, referred to as “PCR”).Such oligonucleotides for a primer utilizing a common feature of PHAsynthase genes include, but are not limited to, a sequence described inTimm, A. and Steinbuchel, A., Eur. J. Biochem., 209, 15 (1992). Anoligonucleotide may be synthesized using, for example, a commerciallyavailable DNA synthesizer such as Custom Synthesis Service,Amersham-Pharmacia Biotech, depending on a designed sequence.

For a PHA synthase gene derived from P161 of this invention, syntheticDNAs having the sequences of SEQ ID NOs. 6 and 7 were designed.

Then, the designed oligonucleotide as a primer is subject to polymerasechain reaction (PCR) using a chromosome DNA in P161 strain as a templateto obtain a PCR amplified fragment. The PCR amplified fragment, which isderived from the primer, comprises a sequence common in PHA synthasegenes at both ends. A partial sequence derived from the PHA synthasegene itself in P161 strain as a template is contained between sequencescomplementary to the primer at both ends.

The PCR amplified fragment obtained is, therefore, almost 100%homologous to the PHA synthase gene in P161 strain and is expected toexhibit a higher S/N ratio as a probe in colony hybridization. Inaddition, it may facilitate stringency control of hybridization.

The above PCR amplified fragment is labeled with an appropriate reagentand used as a probe to colony-hybridize the above chromosome DNA libraryfor selecting a recombinant E. coli strain retaining the PHA synthasegene (Current Protocols in Molecular Biology, Vol. 1, p. 6.0.3 (1994)).For example, the PCR amplified fragment may be labeled using a commondetection system using a labeled enzyme or a commercially available kitsuch as AlkPhosDirect (Amersham-Pharmacia Biotech).

A recombinant E. coli strain retaining a gene fragment comprising a PHAsynthase gene may be selected by, in addition to the above method usinga gene type, a method using a phenotype where PHA synthesis is directlyevaluated. Specifically, in expression of a PHA synthase from a retainedPHA synthase gene in a recombinant E. coli strain, PHA is produced bythe PHA synthase. PHA synthesis may be detected to select a recombinantE. coli strain in which the PHA synthase is expressed. PHA synthesis maybe detected by, for example, staining with Sudan Black B (Archives ofBiotechnology, Vol. 71, p. 283 (1970)) or determination of PHAaccumulation by phase contrast microscopy.

A plasmid is collected from a recombinant E. coli selected by any of theabove methods using an alkali method (Current Protocols in MolecularBiology, Vol. 1, p. 1.6.1 (1994)). The collected plasmid may be used toprovide a DNA fragment comprising a PHA synthase gene or multiple DNAfragments partially containing a PHA synthase gene. The DNA fragmentobtained may be sequenced by, for example, the Sanger's sequencingmethod (Molecular Cloning, Vol. 2, p. 13.3 (1989). Specifically, it maybe conducted by a dye-primer method or a dye-terminator method using anautomatic sequencer such as DNA Sequencer 377A (Perkin Elmer). Since thesequence of the vector itself in which the DNA fragment has beenincorporated is known, the sequence of the DNA fragment cloned thereinmay be unequivocally analyzed.

After sequencing all the obtained DNA fragments comprising a PHAsynthase gene, hybridization may be conducted using a DNA fragmentprepared by an appropriate method such as chemical synthesis, PCR usinga chromosome DNA as a template or degradation of a DNA fragmentcomprising the sequence with a restriction enzyme as a probe to providea PHA synthase gene DNA of this invention.

The inventors have selected a gene translated into a PHA synthaseexhibiting the above substrate specificity from P161 strain according tothe above procedure to find a PHA synthase comprising at least two aminoacid sequences. Specifically, the inventors have found a PHA synthasegene collected from the chromosome DNA of P161 strain and comprising thesequence of SEQ ID NO. 2 and a PHA synthase encoded by the gene andcomprising the amino acid of SEQ ID NO. 1 as well as a PHA synthase genecomprising the sequence of SEQ ID NO. 4 and a PHA synthase encoded bythe gene and comprising the amino acid of SEQ ID NO. 3.

A PHA synthase gene of the present invention may include a degeneratedisomer encoding the same polypeptide which has the same amino acidsequence and is different in a degeneration codon. More specifically, italso includes a degenerated isomer by selection and conversion of a morefrequently used degenerated codon encoding the same amino acid dependingon a host. Besides the PHA synthase comprising the amino acid sequenceof SEQ ID NO. 1 inherent in P161 strain and the PHA synthase comprisingthe amino acid sequence of SEQ ID NO. 3, a PHA synthase of thisinvention may have mutation such as deletion, substitution and additionfor several amino acids as long as its PHA producing activity andsubstrate specificity may not be deteriorated or the amino acid sequencemay be maintained. Mutation such as deletion, substitution and additionmay be introduced by a site mutation introduction technique based on aPHA synthase gene inherent in P161 strain having the sequence of SEQ IDNO. 2 or 4 (Current Protocols in Molecular Biology Vol. 1, p. 8.1.1(1994)).

A recombinant vector of the present invention is used in an applicationwhere a recombinant PHA synthase of this invention is expressed usingPseudomonas sp. or a microorganism such as E. coli as a host. It is,therefore, preferable that the recombinant vector of this inventionitself can autonomously replicate in a host used while comprising apromoter for expression, a PHA synthase gene DNA of this invention and atranscription termination sequence suitable to the host. In addition, itis preferable that after introducing the recombinant vector, a vectorcomprising various marker genes used for its selection is used.

Expression vectors suitable to various types of bacterial hosts such asPseudomonas sp. and E. coli include pLA2917 (ATCC 37355) having a RK2replication origin which may be replicated and retained by a range ofhosts or pJRD215 (ATCC 37533) having a RSF1010 replication origin.Without being limited to these, any vector having a replication originwhich may be replicated and retained by a range of hosts may be used.Any promoter which may be expressed in a bacterium as a host may beused; for example, promoters derived from E. coli, a phage, etc. such astrp, trc, tac, lac, PL, PR, T7 and T3 promoters.

When using a yeast as a host, an expression vector may be YEp13, YCp50,pRS or pYEX vector. A promoter may be, for example, GAL or AOD promoter.

A transformed microorganism of this invention may be produced byintroducing a recombinant vector of this invention into a host suitableto an expression vector used during preparing the recombinant vector.Examples of bacteria which may be used as a host include Esherichia sp.,Pseudomonas sp., Ralstonia sp., Alcaligenes sp., Comamonas sp.,Burkholderia sp., Agrobacterium sp., Flabobacterium sp., Vibrio sp.,Enterobacter sp., Rhizobium sp., Gluconobacter sp., Acinetobacter sp.,Moraxella sp., Nitrosomonas sp., Aeromonas sp., Paracoccus sp., Bacillussp., Clostridium sp., Lactobacillus sp., Corynebacterium sp.,Arthrobacter sp., Achromobacter sp., Micrococcus sp., Mycobacterium sp.,Streptococcus sp., Streptomyces sp., Actinomyces sp., Norcadia sp. andMethylobacterium sp. A recombinant DNA may be introduced into abacterium by an appropriate technique such as the above calcium chloridemethod and electroporation.

Besides the above bacteria, yeasts and molds such as Saccharomyces sp.and Candida sp. may be used as a host. A recombinant DNA may beintroduced into an yeast by, for example, electroporation (MethodsEnzymol., 194, 182-187 (1990)), a spheroplast method (Proc. Natl. Acad.Sci. USA, 84, 1929-1933 (1978)) and a lithium acetate method (J.Bacteriol., 153, 163-168 (1983)).

A PHA synthase of this invention may be prepared by culturing atransformant of this invention prepared by the above procedure andmaking a corresponding PHA synthase gene in an introduced expressionvector producing the synthase as a recombinant protein. The PHA synthaseof this invention is produced and accumulated in the culture (culturedbacterium or culture supernatant) and separated from the culture to beused for production of a recombinant enzyme protein. For this purpose, atransformant of this invention may be cultured by a usual procedure usedfor culturing a host. Culturing may be conducted by any of commonmethods used for culturing a microorganism such as batch, flow batch,continuous culturing and reactor styles. This culturing may be conductedby using, for example, a medium containing an inducer for expressing theabove polyhydroxyalkanoate synthase gene.

For a transformant obtained using a bacterium such as E. coli as a host,a medium used for culturing may be a complete medium or synthetic mediumsuch as LB medium and M9 medium. A microorganism may be grown byaerobically culturing at a culturing temperature of 25 to 37° C. for 8to 72 hours. Then, the bacteria are collected for obtaining a PHAsynthase accumulated in them. Examples of a carbon source for themicroorganism include sugars such as glucose, fructose, sucrose,maltose, galactose and starches; lower alcohols such as ethanol,propanol and butanol; polyalcohols such as glycerol; organic acids suchas acetic acid, citric acid, succinic acid, tartaric acid, lactic acidand gluconic acid; and aliphatic acids such as propionic acid, butanoicacid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid,nonanoic acid, decanoic acid, undecanoic acid and dodecanoic acid.

Examples of a nitrogen source include ammonia; ammonium salts such asammonium chloride, ammonium sulfate and ammonium phosphate; and naturalproduct derivatives such as peptone, meat extract, yeast extract, maltextract, casein decomposition products and corn steep liquor. Examplesof an inorganic material include potassium dihydrogen phosphate,potassium monohydrogen phosphate, magnesium phosphate, magnesium sulfateand sodium chloride. The culture medium may contain an antibiotic suchas kanamycin, ampicillin, tetracyclin, chloramphenicol and streptomycin,depending on, for example, the type of a drug resistance gene used as amarker gene.

When using an inducible promoter in an expression vector, expression maybe enhanced by adding a proper inducer depending on the type of thepromoter during culturing a transformed microorganism. For example, theinducer may be isopropyl-β-D-thiogalactopyranoside (IPTG), tetracyclinor indoleacrylic acid (IAA).

A PHA synthase may be separated and purified by centrifuging andcollecting a culture obtained and processing it by a technique such asaffinity chromatography, cation or anion exchange chromatography and gelfiltration alone or in combination as appropriate. Whether a purifiedmaterial is a desired enzyme is determined by a usual method such as SDSpolyacrylamide gel electrophoresis and Western blotting.

This invention is not limited to the procedures as described above forculturing of a transformed microorganism of this invention, productionof a PHA synthase by the transformed microorganism of this invention andaccumulating it in bacterial cells, and collection and purification ofthe PHA synthase from the cells.

A transformed microorganism of this invention may be used for expressinga recombinant PHA synthase to produce a desired PHA. For example, themicroorganism may be cultured under the above culturing conditions toproduce a recombinant PHA synthase while a substrate corresponding tothe desired PHA on which the PHA synthase acts is added to a medium.Most conveniently, the PHA may be collected from the culture and theproducing bacteria by extraction with an organic solvent commonly usedsuch as chloroform. In an environment where using an organic solventsuch as chloroform is undesirable, the culture may be treated asurfactant such as SDS, an enzyme such as lysozyme, or an agent such asEDTA, sodium hypochlorite and ammonia to remove bacterium componentsother than the PHA for collecting the PHA. This invention is not limitedto the above procedures for culturing of a transformed microorganism ofthis invention for production of a PHA, production of a PHA by andaccumulation thereof in a cultured microorganism, and collection of thePHA from a recombinant microorganism.

EXAMPLES

This invention will be more specifically described with reference toExamples, although these Examples are illustrated as the bestembodiments of this invention and do not limit the technical range ofthis invention.

Example 1 Cloning of a PHA Synthase Gene of P161 Strain

P161 strain was cultured in 100 mL of LB medium (1% polypeptone, 0.5%yeast extract, 0.5% sodium chloride, pH 7.4) at 30° C. overnight andthen a chromosome DNA was separated and collected as described byMarmer. The obtained chromosome DNA was completely digested using arestriction enzyme BglII. A vector pUC18 was cleaved with a restrictionenzyme BamHI. After dephosphorylation of the terminals (MolecularCloning, Vol. 1, p. 5.7.2 (1989), Cold Spring Harbor Laboratory), thecleaved site of the vector (cloning site) and the chromosome DNAfragment after BglII complete digestion were ligated using a DNAligation kit Ver. II (Takara Shuzo Co., Ltd.). The plasmid vector inwhich the chromosome DNA fragment was integrated was used to transformEscherichia coli HB101 for preparing a chromosome DNA library for P161strain.

Then, in order to select a DNA fragment comprising a PHA synthase geneof P161 strain, a probe for colony hybridization was prepared. Anoligonucleotide consisting of the sequences of SEQ ID NOs. 6 and 7(Amersham-Pharmacia Biotech) was prepared and used as a primer for PCRusing the chromosome DNA as a template. A PCR-amplified DNA fragment wasused as a probe. Labeling of the probe was conducted using acommercially available labeling enzyme system AlkPhosDirect(Amersham-Pharmacia Biotech). The labeled probe thus obtained was usedto select an E. coli strain containing a recombinant plasmid comprisingthe PHA synthase gene from the chromosome DNA library of P161 strain bycolony hybridization. From the selected strain, the plasmid wascollected by an alkali method to prepare a DNA fragment comprising a PHAsynthase gene.

The gene DNA fragment thus obtained was recombined in a vector pBBR122(Mo BiTec) comprising a wide host range of replication region which didnot belong to IncP, IncQ or IncW in an incompatible group. Therecombinant plasmid was transformed in Pseudomonas jessenii P161mlstrain (a strain depleted of PHA synthesizing ability) byelectroporation, and then the P161ml strain regained PHA synthesizingability and exhibited complementarity. It demonstrates that the selectedgene DNA fragment comprises a region of a PHA synthase gene translatableinto a PHA synthase in Pseudomonas jessenii P161 ml.

The DNA fragment comprising a PHA synthase gene was sequenced by theSanger's sequencing method. It was thus found that the determinedsequence comprised the sequences of SEQ ID NOs. 2 and 4 each of whichencoded a peptide chain. As described below, it was determined that bothproteins consisting of a peptide chain had enzyme activity and that thesequences of SEQ ID NOs. 2 and 4 were therefore PHA synthase genes.Specifically, it was found that the sequences of SEQ ID NOs. 2 and 4encoded the amino acid sequences of SEQ ID NOs. 1 and 3, respectively,and that a protein comprising one of these amino acid sequences alonecould produce a PHA.

Example 2 Recombination of a PHA Synthase Gene of P161 Strain to anExpression Vector

A PHA synthase gene having the sequence of SEQ ID NO. 2 was PCRed usinga chromosome DNA as a template to reproduce the whole length of a PHAsynthase gene. An oligonucleotide having a sequence which was anupstream primer to the sequence of SEQ ID NO. 2 and had a sequenceupstream of its initiation codon (SEQ ID NO. 8) and an oligonucleotidehaving a sequence which was a downstream primer to the sequence of SEQID NO. 2 and had a sequence downstream of its termination codon (SEQ IDNO. 9) were designed and prepared (Amersham-Pharmacia Biotech). Usingthese oligonucleotides as a primer, PCR was conducted to amplify thewhole length of the PHA synthase gene (LA-PCR kit; Takara Shuzo Co.,Ltd.).

Likewise, a PHA synthase gene having the sequence of SEQ ID NO. 4 wasPCRed using a chromosome DNA as a template to reproduce the whole lengthof a PHA synthase gene. An oligonucleotide having a sequence which wasan upstream primer to the sequence of SEQ ID NO. 4 and had a sequenceupstream of its initiation codon (SEQ ID NO. 10) and an oligonucleotidehaving a sequence which was a downstream primer to the sequence of SEQID NO. 4 and had a sequence downstream of its termination codon (SEQ IDNO. 11) were designed and prepared (Amersham-Pharmacia Biotech). Usingthese oligonucleotides as a primer, PCR was conducted to amplify thewhole length of the PHA synthase gene (LA-PCR kit; Takara Shuzo Co.,Ltd.).

Each of the obtained PCR amplified fragments containing the whole lengthof the PHA synthase gene was completely digested using a restrictionenzyme HindIII. Separately, an expression vector pTrc99A was alsotruncated with a restriction enzyme HindIII and dephosphorylated(Molecular Cloning, Vol. 1, p. 5.7.2 (1989), Cold Spring HarborLaboratory). To the truncated site of the expression vector pTrc99A wasligated the DNA fragment comprising the whole length of the PHA synthasegene from which unnecessary sequences had been removed at both ends,using a DNA ligation kit Ver. II (Takara Shuzo Co., Ltd.).

Using the recombinant plasmids obtained, Escherichia coli HB101 (TakaraShuzo Co., Ltd.) was transformed by a calcium chloride method. Therecombinants were cultured, and the recombinant plasmids were amplifiedand collected individually. The recombinant plasmid retaining the geneDNA of SEQ ID NO. 2 was designated pP161-C1 (derived from SEQ ID NO. 2)while the recombinant plasmid retaining the gene DNA of SEQ ID NO. 4 wasdesignated pP161-C2 (derived from SEQ ID NO. 4).

Example 3 PHA Production (1) Using a PHA Synthase Gene Recombinant E.coli

Using the recombinant plasmids obtained in Example 2, pP161-C1 (derivedfrom SEQ ID NO. 2) and pP161-C2 (derived from SEQ ID NO. 4), anEscherichia coli HB101fB (fadB deficient strain) was transformed by acalcium chloride method to prepare recombinant E. coli strains retainingthe recombinant plasmid, pP161-C1 and pP161-C2 recombinant strains,respectively.

Each of the pP161-C1 and pP161-C2 recombinant strains was inoculated to200 mL of M9 medium containing 0.5% yeast extract and 0.1% FPVA, and themedium was shaken at 37° C. with a rate of 125 strokes/min. After 24hours, the cells were collected by centrifugation, washed once with coldmethanol and lyophilized.

The lyophilized pellet was suspended in 100 ml of chloroform and thesuspension was stirred at 60° C. for 20 hours to extract a PHA. Afterfiltering the extract through a membrane filter with a pore size of 0.45μm, the filtrate was concentrated by rotary evaporation. Then, theconcentrate was re-suspended in cold methanol and the precipitant wascollected and dried in vacuo to provide a PHA. The PHA thus obtained wassubject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-MS, Shimadzu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. Table 1shows together a cell dry weight, a polymer dry weight for a collectedPHA, a polymer yield per a cell (polymer dry weight/cell dry weight) andidentities of monomer units for each strain.

TABLE 1 pP161-C1 pP161-C2 recombinant recombinant strain strain Cell dryweight 900 mg/L 940 mg/L Polymer dry weight  35 mg/L  37 mg/L Polymerdry weight/Cell dry weight 4% 4% Monomer unit composition (area ratio)3-Hydroxybutyric acid 0% 0% 3-Hydroxyvaleric acid 0% 0%3-Hydroxyhexanoic acid 0% 0% 3-Hydroxyheptanoic acid 7% 5%3-Hydroxyoctanoic acid 6% 5% 3-Hydroxynonanoic acid 9% 12% 3-Hydroxydecanoic acid 12%  12%  3-Hydroxy-5-(4-fluorophenyl) valericacid 66%  66% 

These results show that both pP161-C1 and pP161-C2 recombinant strainsproduce, from the substrate 5-(4-fluorophenyl) valeric acid, PHAscomprising a monomer unit represented by formula (I) derived fromcorresponding 3-hydroxy-5-(4-fluorophenyl) valeric acid as a maincomponent. It is, therefore, demonstrated that although the pP161-C1 andpP161-C2 recombinant strains exclusively produce PHA synthases havingthe amino acid sequences of SEQ ID NOs. 1 and 3 translated from the PHAsynthase genes comprising the sequences of SEQ ID NOs. 2 and 4,respectively, both strains similarly convert the substrate5-(4-fluorophenyl) valeric acid into the monomer unit represented byformula (I) derived from corresponding 3-hydroxy-5-(4-fluorophenyl)valeric acid and produce a PHA containing the monomer unit.

Example 4 PHA Production (2) Using a PHA Synthase Gene Recombinant E.coli

Each of the pP161-C1 and pP161-C2 recombinant strains was inoculated to200 mL of M9 medium containing 0.5% yeast extract and 0.2%4-phenoxybutyric acid(PxBA), and the medium was shaken at 37° C. with arate of 125 strokes/min. After 24 hours, the cells were collected bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform and thesuspension was stirred at 60° C. for 20 hours to extract a PHA. Afterfiltering the extract through a membrane filter with a pore size of 0.45μm, the filtrate was concentrated by rotary evaporation. Then, theconcentrate was re-suspended in cold methanol and the precipitant wascollected and dried in vacuo to provide a PHA. The PHA thus obtained wassubject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-MS, Shimadzu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. Table 2shows together a cell dry weight, a polymer dry weight for a collectedPHA, a polymer yield per a cell (polymer dry weight/cell dry weight) andidentities of monomer units for each strain.

TABLE 2 pP161-C1 pP161-C2 recombinant recombinant strain strain Cell dryweight 750 mg/L 720 mg/L Polymer dry weight  4 mg/L  4 mg/L Polymer dryweight/Cell dry weight 0.5%   0.5%   Monomer unit composition (arearatio) 3-Hydroxybutyric acid 0% 0% 3-Hydroxyvaleric acid 0% 0%3-Hydroxyhexanoic acid 0% 0% 3-Hydroxyheptanoic acid 2% 2%3-Hydroxyoctanoic acid 3% 3% 3-Hydroxynonanoic acid 5% 7%3-Hydroxydecanoic acid 5% 6% 3-Hydroxy-4-phenoxybutyric acid 85%  82% 

These results show that both pP161-C1 and pP161-C2 recombinant strainsproduce, from the substrate 4-phenoxybutyric acid, PHAs comprising amonomer unit represented by formula (VI) derived from corresponding3-hydroxy-4-phenoxybutyric acid as a main component. It is, therefore,demonstrated that although the pP161-C1 and pP161-C2 recombinant strainsexclusively produce PHA synthases having the amino acid sequences of SEQID NOs. 1 and 3 translated from the PHA synthase genes comprising thesequences of SEQ ID NOs. 2 and 4, respectively, both strains similarlyconvert the substrate 4-phenoxybutyric acid into the monomer unitrepresented by formula (VI) derived from corresponding3-hydroxy-4-phenoxybutyric acid and produce a PHA containing the monomerunit.

Example 5 PHA Production (3) Using a PHA Synthase Gene Recombinant E.coli

Each of the pP161-C1 and pP161-C2 recombinant strains was inoculated to200 mL of M9 medium containing 0.5% yeast extract and 0.1%6-phenylhexanoic acid (PHxA), and the medium was shaken at 37° C. with arate of 125 strokes/min. After 24 hours, the cells were collected bycentrifugation, washed once with cold methanol and lyophilized.

The lyophilized pellet was suspended in 100 mL of chloroform and thesuspension was stirred at 60° C. for 20 hours to extract a PHA. Afterfiltering the extract through a membrane filter with a pore size of 0.45μm, the filtrate was concentrated by rotary evaporation. Then, theconcentrate was re-suspended in cold methanol and the precipitant wascollected and dried in vacuo to provide a PHA. The PHA thus obtained wassubject to methanolysis as usual and analyzed using a gaschromatography-mass spectrometry apparatus (GC-MS, Shimadzu QP-5050, EItechnique) to identify methyl-esterified PHA monomer units. Table 3shows together a cell dry weight, a polymer dry weight for a collectedPHA, a polymer yield per a cell (polymer dry weight/cell dry weight) andidentities of monomer units for each strain.

TABLE 3 pP161-C1 pP161-C2 recombinant recombinant strain strain Cell dryweight 1050 mg/L 980 mg/L Polymer dry weight  73 mg/L  70 mg/L Polymerdry weight/Cell dry weight 7% 7% Monomer unit composition (area ratio)3-Hydroxybutyric acid 0% 0% 3-Hydroxyvaleric acid 0% 0%3-Hydroxyhexanoic acid 0% 0% 3-Hydroxyheptanoic acid 3% 3%3-Hydroxyoctanoic acid 3% 4% 3-Hydroxynonanoic acid 5% 2%3-Hydroxydecanoic acid 5% 4% 3-Hydroxy-6-phenylhexanoic acid 84%  87% 

These results show that both pP161-C1 and pP161-C2 recombinant strainsproduce, from the substrate 6-phenylhexanoic acid, PHAs comprising amonomer unitrepresented by formula (IX) derived from corresponding3-hydroxy-6-phenylhexanoic acid as a main component. It is, therefore,demonstrated that although the pP161-C1 and pP161-C2 recombinant strainsexclusively produce PHA syntheses having the amino acid sequences of SEQID NOs. 1 and 3 translated from the PHA synthase genes comprising thesequences of SEQ ID NOs. 2 and 4, respectively, both strains similarlyconvert the substrate 6-phenylhexanoic acid into the monomer unitrepresented by formula (IX) derived from corresponding3-hydroxy-6-phenylhexanoic acid and produce a PHA containing the monomerunit.

The results together with those in Examples 3 and 4 demonstrate that thePHA synthases having the amino acid sequences of SEQ ID NOs. 1 and 3have enzyme activity mutually similar in substrate specificity.

Example 6 Homology of a PHA Synthase Gene of P161 Strain

In the same manner as in Example 1, the chromosome DNA of P161 strainwas separated and collected. Further, Pseudomonas olevorans ATCC29347,Pseudomonas putida Tk2440 and Pseudomonas aeruginose PA01 were culturedand the chromosome DNAs thereof were separated and collected in the sameas in Example 1.

Next, a probe for hybridization was prepared for confirming homology ofa PHAsynthase gene of P161 strain. In the same manner as in Example 2,an oligonucleotide having the sequences of SEQ ID NOs 8 and 9 weresynthesized. (Amersham-Pharmacia Biotech) PCR was conducted using thethus synthesized oligonucleotide as a primer and the chromosome DNA as atemplate. The obtained PCR amplified DNA fragments were used as a probe.Labeling of the probe using AlkphosDirect (Amersham-Pharmacia Biotech)was conducted to obtain the labeled probe referred to as “phaC1”. In thesame manner as above, an oligonucleotide having the sequences of SEQ IDNOs 10 and 11 were synthesized. (Amersham-Pharmacia Biotech) PCR wasconducted using the thus synthesized oligonucleotide as a primer and thechromosome DNA as a template. Labeling of the obtained PCR amplified DNAfragments was conducted in the same manner as above to obtain thelabeled probe referred to as “phaC2”.

Using the thus obtained probes, homology of the PHA synthase gene wasconfirmed by a dot-blot method. The thus prepared chromosome DNA wasalkalized and then blotted on a nylon film (Tropilon-45, produced byTropix Co.) by 1 μg at respective portions using a dot-blot apparatus(BRL).

The film was dried at 80° C. for two hours, then put in a vinyl bag. 3ml of the solution for hybridization prepared according to the recipe ofAlkPhosDirect was added thereto, and hybridization was conducted at 55°C. for one hour. 15 ng of the labeled probe phaC1 or phaC2 per 3 ml ofthe above hybridization solution (5 ng/ml) was added to the nylon film,and hybridization was conducted at 55° C. for 12 hours. And then thenylon film was put out from the vinyl bag, and washed two times for 10minutes at 55° C. with a first washing buffer according the recipe ofAlkPhosDirect. And then after it was washed two times for 5 minutes atroom temperature with a second washing buffer according to the recipe ofAlkPhosDirect, a detecting step was carried out using CDP-Star attachedto AlkPhosDirect according to the recipe.

Further, a detecting step was carried out under the same conditions asabove except that hybridization and first washing were conducted at 60°C. or 65° C. The results were shown in Table 4.

TABLE 4 Temperature (° C.) 55 60 65 Probe phaC1 Target DNA P161 Strain AA B ATCC29347 Strain C D D KT2440 Strain C D D PAO1 Strain D D D ProbephaC2 Target DNA P91 Strain A A B ATCC29347 Strain C D D KT2440 Strain CD D PAO1 Strain D D D A: Strong Signal, B: Signal, C: Slight Signal, D:No Signal

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 11 <210> SEQ ID NO 1 <211> LENGTH: 559<212> TYPE: PRT <213> ORGANISM: Pseudomonas jessenii P161 ; FERM #BP-7376 <400> SEQUENCE: 1 Met Ser Asn Lys Asn Asn Asp Asp Leu Lys Se#r Gln Ala Ser Glu   1               5  #                 10 #                 15 Asn Thr Leu Gly Leu Asn Pro Val Val Gly Le#u Arg Gly Lys Asp                  20  #                 25 #                 30 Leu Leu Ala Ser Ala Arg Met Val Leu Arg Gl#n Ala Ile Lys Gln                  35  #                 40 #                 45 Pro Ile His Ser Ala Arg His Val Ala His Ph#e Gly Leu Glu Leu                  50  #                 55 #                 60 Lys Asn Val Leu Leu Gly Lys Ser Glu Leu Le#u Pro Thr Ser Asp                  65  #                 70 #                 75 Asp Arg Arg Phe Ala Asp Pro Ala Trp Ser Gl#n Asn Pro Leu Tyr                  80  #                 85 #                 90 Lys Arg Tyr Leu Gln Thr Tyr Leu Ala Trp Ar#g Lys Glu Leu His                  95  #                100 #                105 Asp Trp Ile Asp Asp Ser Asn Leu Pro Ala Ly#s Asp Val Ser Arg                 110   #               115  #               120 Gly His Phe Val Ile Asn Leu Met Thr Glu Al#a Phe Ala Pro Thr                 125   #               130  #               135 Asn Thr Ala Ala Asn Pro Ala Ala Val Lys Ar#g Phe Phe Glu Thr                 140   #               145  #               150 Gly Gly Lys Ser Leu Leu Asp Gly Leu Ser Hi#s Leu Ala Lys Asp                 155   #               160  #               165 Leu Val His Asn Gly Gly Met Pro Ser Gln Va#l Asn Met Gly Ala                 170   #               175  #               180 Phe Glu Val Gly Lys Thr Leu Gly Val Thr Gl#u Gly Ala Val Val                 185   #               190  #               195 Phe Arg Asn Asp Val Leu Glu Leu Ile Gln Ty#r Lys Pro Ile Thr                 200   #               205  #               210 Glu Gln Val His Glu Arg Pro Leu Leu Val Va#l Pro Pro Gln Ile                 215   #               220  #               225 Asn Lys Phe Tyr Val Phe Asp Leu Ser Pro Gl#u Lys Ser Leu Ala                 230   #               235  #               240 Arg Phe Cys Leu Arg Asn Asn Val Gln Thr Ph#e Ile Val Ser Trp                 245   #               250  #               255 Arg Asn Pro Thr Lys Glu Gln Arg Glu Trp Gl#y Leu Ser Thr Tyr                 260   #               265  #               270 Ile Glu Ala Leu Lys Glu Ala Val Asp Val Va#l Thr Ala Ile Thr                 275   #               280  #               285 Gly Ser Lys Asp Val Asn Met Leu Gly Ala Cy#s Ser Gly Gly Ile                 290   #               295  #               300 Thr Cys Thr Ala Leu Leu Gly His Tyr Ala Al#a Ile Gly Glu Asn                 305   #               310  #               315 Lys Val Asn Ala Leu Thr Leu Leu Val Ser Va#l Leu Asp Thr Thr                 320   #               325  #               330 Leu Asp Ser Asp Val Ala Leu Phe Val Asp Gl#u Gln Thr Leu Glu                 335   #               340  #               345 Ala Ala Lys Arg Gln Ser Tyr Gln Ala Gly Va#l Leu Glu Gly Arg                 350   #               355  #               360 Asp Met Ala Lys Val Phe Ala Trp Met Arg Pr#o Asn Asp Leu Ile                 365   #               370  #               375 Trp Asn Tyr Trp Val Asn Asn Tyr Leu Leu Gl#y Asn Glu Pro Pro                 380   #               385  #               390 Val Phe Asp Ile Leu Phe Trp Asn Asn Asp Th#r Thr Arg Leu Pro                 395   #               400  #               405 Ala Ala Phe His Gly Asp Leu Ile Glu Met Ph#e Lys Ser Asn Pro                 410   #               415  #               420 Leu Thr Arg Ala Asp Ala Leu Glu Val Cys Gl#y Thr Pro Ile Asp                 425   #               430  #               435 Leu Lys Lys Val Thr Ala Asp Ile Phe Ser Le#u Ala Gly Thr Ser                 440   #               445  #               450 Asp His Ile Thr Pro Trp Arg Ser Cys Tyr Ly#s Ser Ala Gln Leu                 455   #               460  #               465 Phe Gly Gly Asn Val Glu Phe Val Leu Ser Se#r Ser Gly His Ile                 470   #               475  #               480 Gln Ser Ile Leu Asn Pro Pro Gly Asn Pro Ly#s Ser Arg Tyr Met                 485   #               490  #               495 Thr Ser Thr Glu Met Pro Ala Asn Ala Asp As#p Trp Gln Glu Glu                 500   #               505  #               510 Ser Thr Lys His Ala Asp Ser Trp Trp Leu Hi#s Trp Gln Ala Trp                 515   #               520  #               525 Gln Ala Gln Arg Ser Gly Asn Leu Lys Lys Al#a Pro Leu Lys Leu                 530   #               535  #               540 Gly Asn Lys Ala Tyr Pro Ala Gly Glu Ala Al#a Pro Gly Thr Tyr                 545   #               550  #               555 Val His Glu Arg <210> SEQ ID NO 2 <211> LENGTH: 1680<212> TYPE: DNA <213> ORGANISM: Pseudomonas jessenii P161 ; BP-737 #6<400> SEQUENCE: 2atgagtaaca agaataacga tgacttgaag agtcaagcct cggaaaacac  #              50cttggggctg aatcctgtcg ttggactgcg tggaaaggat ctactggctt  #             100ctgctcgaat ggtgctcagg caggccatca agcaaccgat tcacagcgcc  #             150aggcatgtcg ctcatttcgg cctggaactc aagaacgtgc tgctcggcaa  #             200atccgagctg ctaccgacca gcgatgaccg tcgtttcgcg gatccggcct  #             250ggagccagaa cccgctctac aaacgttatc tgcaaaccta cctggcgtgg  #             300cgcaaggaac tccacgactg gatcgacgac agcaacctgc cggccaagga  #             350cgtcagccgc gggcacttcg tgatcaacct catgaccgag gccttcgccc  #             400cgaccaacac ggcggccaac ccggcggcgg tcaaacgctt cttcgaaacc  #             450ggtggcaaga gcctgctcga tggcctctcg catctggcca aggacctggt  #             500acataacggc ggcatgccga gccaggtcaa catgggcgca ttcgaggtcg  #             550gcaagaccct tggcgtgacc gagggcgcgg tggtctttcg caatgacgtg  #             600ctggaactga tccagtacaa accgatcacc gagcaggtgc atgaacgccc  #             650actgctggtg gtaccgccac agatcaacaa gttctacgtt ttcgacctga  #             700gcccggaaaa gagcctggcg cgattctgcc tgcgcaacaa cgtgcagacc  #             750ttcatcgtca gctggcgcaa cccgaccaag gagcagcgcg agtggggcct  #             800gtcgacctac atcgaagcgc tcaaggaagc ggttgatgtg gtcaccgcca  #             850tcaccggcag caaagacgtg aacatgctcg gtgcctgctc cggcggcatc  #             900acctgcaccg cgctgctggg ccactacgca gcaatcggcg agaacaaggt  #             950caacgccctg accctgctgg tcagcgtgct cgacaccacc ctggacagcg  #            1000acgtggccct gttcgtcgac gagcagaccc tcgaagccgc caagcgccag  #            1050tcgtaccagg ccggtgtact cgaaggccgt gacatggcga aagtcttcgc  #            1100ctggatgcgc cccaacgacc tgatctggaa ctactgggtc aacaactact  #            1150tgttgggcaa cgagccgccg gtattcgaca ttctgttctg gaacaacgac  #            1200accacccggt tgcccgccgc gttccatggc gacctgatcg agatgttcaa  #            1250aagtaacccg ttgacccgtg ccgatgcact ggaagtgtgc ggtacgccga  #            1300tcgatctgaa gaaagtcacc gccgacatct tctcgctggc cggcaccagc  #            1350gaccacatta ccccgtggcg ctcctgctac aagtcggcgc aactgttcgg  #            1400cggcaacgtt gaattcgtat tgtccagcag cgggcacatc cagagcattc  #            1450tgaacccgcc gggcaatccg aaatcgcgtt acatgaccag caccgaaatg  #            1500cccgccaatg ccgatgactg gcaggaagag tcgaccaagc acgccgactc  #            1550ctggtggctg cactggcagg catggcaggc acagcgttcg ggcaacctga  #            1600aaaaagcccc gctgaaattg ggcaacaagg cctatccagc gggtgaagcc  #            1650 gcaccgggca cttacgtgca tgagcggtaa         #                   #         1680 <210> SEQ ID NO 3 <211> LENGTH: 560<212> TYPE: PRT <213> ORGANISM: Pseudomonas jessenii P161 ; BP-737 #6<400> SEQUENCE: 3 Met Arg Glu Lys Pro Ala Arg Asp Ser Leu Pr#o Thr Pro Ala Ala   1               5  #                 10 #                 15 Phe Ile Asn Ala Gln Ser Ala Ile Thr Gly Le#u Arg Gly Arg Asp                  20  #                 25 #                 30 Leu Leu Ser Thr Leu Arg Ser Val Ala Ala Hi#s Gly Leu Arg Asn                  35  #                 40 #                 45 Pro Val His Ser Ala Arg His Ala Leu Lys Le#u Gly Gly Gln Leu                  50  #                 55 #                 60 Gly Arg Val Leu Leu Gly Glu Thr Leu His Pr#o Thr Asn Pro Gln                  65  #                 70 #                 75 Asp Thr Arg Phe Ala Asp Pro Ala Trp Ser Le#u Asn Pro Phe Tyr                  80  #                 85 #                 90 Arg Arg Ser Leu Gln Ala Tyr Leu Ser Trp Gl#n Lys Gln Val Lys                  95  #                100 #                105 Ser Trp Ile Asp Glu Ser Asn Met Ser Pro As#p Asp Arg Ala Arg                 110   #               115  #               120 Ala His Phe Ala Phe Ala Leu Leu Asn Asp Al#a Val Ser Pro Ser                 125   #               130  #               135 Asn Thr Leu Leu Asn Pro Leu Ala Val Lys Gl#u Phe Phe Asn Ser                 140   #               145  #               150 Gly Gly Asn Ser Leu Val Arg Gly Ile Gly Hi#s Leu Val Asp Asp                 155   #               160  #               165 Leu Leu His Asn Asp Gly Leu Pro Arg Gln Va#l Thr Lys Gln Ala                 170   #               175  #               180 Phe Glu Val Gly Lys Thr Val Ala Thr Thr Th#r Gly Ala Val Val                 185   #               190  #               195 Phe Arg Asn Glu Leu Leu Glu Leu Ile Gln Ty#r Lys Pro Met Ser                 200   #               205  #               210 Glu Lys Gln Tyr Ser Lys Pro Leu Leu Val Va#l Pro Pro Gln Ile                 215   #               220  #               225 Asn Lys Tyr Tyr Ile Phe Asp Leu Ser Pro Hi#s Asn Ser Phe Val                 230   #               235  #               240 Gln Tyr Ala Leu Lys Asn Gly Leu Gln Thr Ph#e Met Ile Ser Trp                 245   #               250  #               255 Arg Asn Pro Asp Val Arg His Arg Glu Trp Gl#y Leu Ser Thr Tyr                 260   #               265  #               270 Val Glu Ala Val Glu Glu Ala Met Asn Val Cy#s Arg Ala Ile Thr                 275   #               280  #               285 Gly Ala Arg Glu Val Asn Leu Met Gly Ala Cy#s Ala Gly Gly Leu                 290   #               295  #               300 Thr Ile Ala Ala Leu Gln Gly His Leu Gln Al#a Lys Arg Gln Leu                 305   #               310  #               315 Arg Arg Val Ser Ser Ala Thr Tyr Leu Val Se#r Leu Leu Asp Ser                 320   #               325  #               330 Glu Leu Asp Ser Pro Ala Ser Leu Phe Ala As#p Glu Gln Thr Leu                 335   #               340  #               345 Glu Ala Ala Lys Arg Arg Ser Tyr Gln Lys Gl#y Val Leu Asp Gly                 350   #               355  #               360 Arg Asp Met Ala Lys Val Phe Ala Trp Met Ar#g Pro Asn Asp Leu                 365   #               370  #               375 Ile Trp Ser Tyr Phe Val Asn Asn Tyr Leu Le#u Gly Lys Glu Pro                 380   #               385  #               390 Pro Ala Phe Asp Ile Leu Tyr Trp Asn Asn As#p Ser Thr Arg Leu                 395   #               400  #               405 Pro Ala Ala Leu His Gly Asp Leu Leu Asp Ph#e Phe Lys His Asn                 410   #               415  #               420 Pro Leu Thr His Pro Gly Gly Leu Glu Val Cy#s Gly Thr Pro Ile                 425   #               430  #               435 Asp Leu Gln Lys Val Thr Val Asp Ser Phe Se#r Val Ala Gly Ile                 440   #               445  #               450 Asn Asp His Ile Thr Pro Trp Asp Ala Val Ty#r Arg Ser Ala Leu                 455   #               460  #               465 Leu Leu Gly Gly Glu Arg Arg Phe Val Leu Se#r Asn Ser Gly His                 470   #               475  #               480 Val Gln Ser Ile Leu Asn Pro Pro Ser Asn Pr#o Lys Ala Asn Tyr                 485   #               490  #               495 Val Glu Asn Gly Lys Leu Ser Ser Asp Pro Ar#g Ala Trp Tyr Tyr                 500   #               505  #               510 Asp Ala Arg His Val Asp Gly Ser Trp Trp Th#r Gln Trp Leu Ser                 515   #               520  #               525 Trp Ile Gln Glu Arg Ser Gly Ala Gln Lys Gl#u Thr His Met Ala                 530   #               535  #               540 Leu Gly Asn Gln Asn Tyr Pro Pro Met Glu Al#a Ala Pro Gly Thr                 545   #               550  #               555 Tyr Val Arg Val Arg                 560<210> SEQ ID NO 4 <211> LENGTH: 1683 <212> TYPE: DNA<213> ORGANISM: Pseudomonas jessenii P161 ; BP-737 #6 <400> SEQUENCE: 4atgcgcgaga aaccagcgag ggattcctta ccgactcccg ccgcgttcat  #              50caatgcacag agtgcgatta ccggcctgcg cggtcgggat ctgttatcga  #             100ccctgcgtag tgtggccgcc catggcttgc gcaatccggt gcacagtgcc  #             150cgacatgccc tcaaactcgg cggccagctc ggtcgtgtgt tgctgggcga  #             200aaccctgcac ccgaccaacc cgcaggacac tcgcttcgcc gatccggcgt  #             250ggagcctcaa cccgttttat cggcgcagcc tgcaggctta tctgagctgg  #             300cagaagcagg tcaaaagctg gatcgacgag agcaacatga gcccggacga  #             350ccgtgcccgc gcccacttcg ctttcgcctt gctcaacgac gccgtatcgc  #             400cctccaacac cctgctcaat ccattggcgg tcaaggagtt cttcaattcc  #             450gggggtaaca gcctggtgcg tggcatcggc catctggtgg acgatctgct  #             500gcacaacgat ggcctgcccc ggcaagtcac caagcaagcg ttcgaggtcg  #             550gcaagacggt cgccaccacc accggtgccg tggtgtttcg caacgaactg  #             600ctggagttga tccagtacaa gccgatgagc gaaaagcagt attccaagcc  #             650cctgttggtg gtgccgccgc aaatcaacaa gtactacatt ttcgacctga  #             700gcccccacaa cagcttcgtc cagtacgcgc tgaaaaacgg cctgcaaacc  #             750ttcatgatca gctggcgcaa cccggatgtg cgtcaccgcg aatgggggct  #             800ctcgacctac gtggaagccg tggaagaagc catgaatgtc tgccgggcga  #             850tcaccggtgc acgcgaggtc aacctgatgg gcgcctgcgc cggcgggctg  #             900accattgccg cgttgcaggg ccacttgcaa gccaaacggc agctgcgcag  #             950ggtgtccagt gcaacgtatc tggtgagcct gctcgacagt gaactggaca  #            1000gccccgcttc actgttcgcc gacgaacaga ctctggaggc tgccaagcgt  #            1050cgctcctatc agaaaggtgt gctggacggc cgcgacatgg ccaaggtctt  #            1100cgcctggatg cgccccaacg atttgatctg gagctacttc gtcaacaact  #            1150acctgttggg caaggagccg ccggcgttcg acatcctcta ctggaacaac  #            1200gacagcacgc gcttgcctgc cgccctgcat ggcgacctgc tggacttctt  #            1250caagcacaac ccgctgaccc acccgggcgg cctggaagtg tgtggcacgc  #            1300cgatcgattt gcagaaggtc accgttgaca gcttcagcgt cgccggcatc  #            1350aacgatcaca tcacgccttg ggatgcggtg tatcgctcgg cgctgttgct  #            1400cggtggcgag cggcgcttcg tgctgtccaa cagcggccat gtgcagagca  #            1450tcctcaaccc gccgagcaac ccgaaagcca actacgtcga aaacggcaag  #            1500ctgagcagcg acccccgcgc ctggtactac gacgccaggc atgtcgacgg  #            1550cagttggtgg acccaatggc tgagctggat tcaggaacgc tccggcgcgc  #            1600agaaggaaac ccacatggcg ctcggcaacc agaactatcc accgatggaa  #            1650 gctgcgcccg gtacctacgt acgtgtgcgc tga       #                   #       1683 <210> SEQ ID NO 5 <211> LENGTH: 1501<212> TYPE: DNA <213> ORGANISM: Pseudomonas jessenii P161 ; BP-737 #6<220> FEATURE: <220> FEATURE: cDNA to 16S rRNA <400> SEQUENCE: 5tgaacgctgg cggcaggcct aacacatgca agtcgagcgg atgacgggag  #              50cttgctcctg aattcagcgg cggacgggtg agtaatgcct aggaatctgc  #             100ctggtagtgg gggacaacgt ctcgaaaggg acgctaatac cgcatacgtc  #             150ctacgggaga aagcagggga ccttcgggcc ttgcgctatc agatgagcct  #             200aggtcggatt agctagttgg tgaggtaatg gctcaccaag gcgacgatcc  #             250gtaactggtc tgagaggatg atcagtcaca ctggaactga gacacggtcc  #             300agactcctac gggaggcagc agtggggaat attggacaat gggcgaaagc  #             350ctgatccagc catgccgcgt gtgtgaagaa ggtcttcgga ttgtaaagca  #             400ctttaagttg ggaggaaggg cattaaccta atacgttagt gttttgacgt  #             450taccgacaga ataagcaccg gctaactctg tgccagcagc cgcggtaata  #             500cagagggtgc aagcgttaat cggaattact gggcgtaaag cgcgcgtagg  #             550tggtttgtta agttggatgt gaaagccccg ggctcaacct gggaactgca  #             600ttcaaaactg acaagctaga gtatggtaga gggtggtgga atttcctgtg  #             650tagcggtgaa atgcgtagat ataggaagga acaccagtgg cgaaggcgac  #             700cacctggact gatactgaca ctgaggtgcg aaagcgtggg gagcaaacag  #             750gattagatac cctggtagtc cacgccgtaa acgatgtcaa ctagccgttg  #             800ggagccttga gctcttagtg gcgcagctaa cgcattaagt tgaccgcctg  #             850gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca  #             900caagcggtgg agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc  #             950aggccttgac atccaatgaa ctttccagag atggatgggt gccttcggga  #            1000acattgagac aggtgctgca tggctgtcgt cagctcgtgt cgtgagatgt  #            1050tgggttaagt cccgtaacga gcgcaaccct tgtccttagt taccagcacg  #            1100taatggtggg cactctaagg agactgccgg tgacaaaccg gaggaaggtg  #            1150gggatgacgt caagtcatca tggcccttac ggcctgggct acacacgtgc  #            1200tacaatggtc ggtacagagg gttgccaagc cgcgaggtgg agctaatccc  #            1250acaaaaccga tcgtagtccg gatcgcagtc tgcaactcga ctgcgtgaag  #            1300tcggaatcgc tagtaatcgc gaatcagaat gtcgcggtga atacgttccc  #            1350gggccttgta cacaccgccc gtcacaccat gggagtgggt tgcaccagaa  #            1400gtagctagtc taaccttcgg gaggacggtt accacggtgt gattcatgac  #            1450tggggtgaag tcgtaccaag gtagccgtag gggaacctgc ggctggatca  #            1500 c                   #                  #                   #             1501 <210> SEQ ID NO 6<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer for PCR multiplica #tion<400> SEQUENCE: 6 tgctggaact gatccagtac             #                  #                   # 20 <210> SEQ ID NO 7 <211> LENGTH: 23<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer for PCR multiplica #tion<400> SEQUENCE: 7 gggttgagga tgctctggat gtg           #                   #                23 <210> SEQ ID NO 8<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Primer for PCR multiplica #tion<400> SEQUENCE: 8 ctacaaagct tgacccggta ctcgtctcag         #                   #           30 <210> SEQ ID NO 9 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer for PCR multiplica #tion<400> SEQUENCE: 9 gcgagcaagc ttgctcctac agggatagc         #                   #            29 <210> SEQ ID NO 10 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer for PCR multiplica #tion<400> SEQUENCE: 10 gttttaagct tgaagacgaa ggagtgttg         #                   #            29 <210> SEQ ID NO 11 <211> LENGTH: 30<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Primer for PCR multiplica #tion<400> SEQUENCE: 11 ctcctacaag cttggagact gactgtggcc         #                   #            30

What is claimed is:
 1. An isolated polyhydroxyalkanoate synthase havingthe amino acid sequence of SEQ ID NO:
 3. 2. An isolatedpolyhydroxyalkanoate synthase having an amino acid sequence, which is atleast 95% homologous with the amino acid sequence of SEQ ID NO:
 3. 3. Amethod for preparing the polyhydroxyalkanoate synthase according toclaim 1, comprising the steps of: culturing the transformedmicroorganism harboring a recombinant vector containing a DNA sequenceencoding the polyhydroxyalkanoate synthase in a medium; expressing saidDNA; and isolating the polyhydroxyalkanoate synthase from the cultureobtained.
 4. A method for preparing the polyhydroxyalkanoate synthaseaccording to claim 2, comprising the steps of: culturing the transformedmicroorganism harboring a recombinant vector containing a DNA sequenceencoding the polyhydroxyalkanoate synthase in a medium; expressing saidDNA; and isolating the polyhydroxyalkanoate synthase from the cultureobtained.
 5. The method according to claim 3, wherein the DNA has thesequence according to SEQ ID NO:
 4. 6. The method according to claim 4,wherein the DNA has the sequence according to SEQ ID NO: 4.